Hydrophilic high quantum yield acridinium esters with improved stability and fast light emission

ABSTRACT

Hydrophilic, high quantum yield, chemiluminescent acridinium compounds with increased light output, improved stability, fast light emission and decreased non specific binding are disclosed. The chemiluminescent acridinium esters possess hydrophilic, branched, electron-donating functional groups at the C2 and/or C7 positions of the acridinium nucleus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/843,528 filed Jul. 8, 2013, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to hydrophilic, high quantum yield,chemiluminescent acridinium compounds with increased light output,improved stability, fast light emission and low non-specific binding.These compounds because of their enhanced quantum yield and hydrophilicnature, are useful in improving assay sensitivity. The improvedstability of these compounds is useful for extending the shelf life ofreagents using these compounds as well as for minimizing variation inassay performance with time. Their increased emission kinetics alsopermits faster light measurements in assays especially in automatedanalyzers.

BACKGROUND OF THE INVENTION

Chemiluminescent acridinium esters (AEs) are extremely useful labelsthat have been used extensively in immunoassays and nucleic acid assays.A review by Pringle, M. J. Journal of Clinical Ligand Assay vol. 22, pp.105-122 (1999) summarizes past and current developments in this class ofchemiluminescent compounds.

McCapra, F. et al. in Tetrahedron Lett. vol. 43, pp.3167-3172 (1964) andRahut et al. in J. Org. Chem vol. 301, pp. 3587-3592. (1965) disclosedthat chemiluminescence from the esters of acridinium salts can betriggered by alkaline peroxide. Since these seminal studies, interest inacridinium compounds has increased because of their utility as labels.The application of the acridinium ester9-carboxyphenyl-N-methylacridinium bromide in an immunoassay wasdisclosed by Simpson, J. S. A. et al., Nature, vol. 279, pp. 646-647(1979). However, this acridinium ester is quite unstable, therebylimiting its commercial utility. This instability arises from hydrolysisof the 9-carboxyphenyl ester linkage between the phenol and theacridinium ring.

Different strategies for increasing the stability of acridiniumcompounds have been described. Law et al., Journal of Bioluminescenceand Chemiluminescence, vol. 4, pp. 88-89 (1989), introduced two methylgroups to flank the acridinium ester moiety to stabilize this linkage.The resulting sterically stabilized acridinium ester, DMAE-NHS[2′,6′-dimethyl-4′-(N-succinimidyloxycarbonyl)phenyl10-methylacridinium-9-carboxylate] was found to have the same lightoutput as an acridinium ester lacking the two methyl groups. Thestability of the former compound when conjugated to an immunoglobulinwas vastly superior and showed no loss of chemiluminescent activity evenafter one week at 37° C. at pH 7. In contrast, the unsubstitutedacridinium ester only retained 10% of its activity when subjected to thesame treatment. U.S. Pat. Nos. 4,918,192 and 5,110.932 describe DMAE andits applications.

U.S. Pat. No. 5,656,426 to Law et al. discloses a hydrophilic version ofDMAE termed NSP-DMAE-NHS ester where the N-methyl group has beenreplaced with an N-sulfopropyl (NSP) group. The structures of these twocompounds and the numbering system of the acridinium ring areillustrated below.

Natrajan et al. in U.S. Pat. No. 6,664,043 B2 disclosed NSP-DMAEderivatives with hydrophilic modifiers attached to the phenol. Thestructure of one such compound, NSP-DMAE-HEG-Glutarate-NHS, (abbreviatedas HEG-AE) is illustrated in the above. In this compound a diaminohexa(ethylene) glycol (diamino-HEG) moiety is attached to the phenol toincrease the aqueous solubility of the acridinium ester. A glutaratemoiety was appended to the end of HEG and was converted to the NHS esterto enable labeling of various molecules.

A different class of stable chemiluminescent acridinium compounds hasbeen described by Kinkel et al., Journal of Bioluminescence andChemiluminescence vol. 4, pp. 136-139 (1989) and Mattingly, Journal ofBioluminescence and Chemiluminescence vol. 6, pp. 107-114 (1991) andU.S. Pat. No. 5,468,646. In this class of compounds, the phenolic esterlinkage is replaced by a sulfonamide moiety, which is reported to imparthydrolytic stability without compromising the light output. Inacridinium esters, the phenol is the ‘leaving group’ whereas inacridinium sulfonamides, the sulfonamide is the ‘leaving group’ duringthe chemiluminescent reaction with alkaline peroxide.

Light emission from acridinium compounds is normally triggered byalkaline peroxide. The overall light output, which can also be referredto as the chemiluminescence quantum yield, is a combination of theefficiencies of the chemical reaction leading to the formation of theexcited-state acridone and the latter's fluorescence quantum yield.

Recently, Natrajan et al. in U.S. Pat. No. 7,309,615 B2, the disclosureof which is hereby incorporated by reference herein, describedhydrophilic, high quantum yield acridinium compounds containinghydrophilic alkoxy groups (OR*) at C2 and/or C7 of the acridinium ring,wherein R* is a group comprising a sulfopropyl moiety or ethylene glycolmoieties or a combination thereof. The enhanced light output from suchcompounds and their hydrophilic nature made them useful for improvingthe sensitivity of immunoassays. The structure of one such compound,NSP-2,7-(OMHEG)₂-DMAE-AC-NHS (abbreviated as HQYAE), is illustratedbelow.

SUMMARY OF INVENTION

It has surprisingly been found that hydrophilic, high quantum yield,chemiluminescent acridinium esters possessing electron-donatingfunctional groups of the form —OG at C2 and/or C7 of the acridiniumring, where G represents a branched hydrophilic substituent, provideincreased light output, improved stability, fast light emission and/orlow non-specific binding in assays.

In one aspect of the invention, hydrophilic, high quantum yieldacridinium esters are provided having the structure of formula (I):

wherein, R₁ is a methyl or sulfopropyl group; G is a branched groupindependently selected at each occurrence from:

where R₂, R₃, R₄, R₅, R₆ and R₇ are independently at each occurrence amethyl group or a group —(CH₂CH₂O)_(n)CH₃, where n is an integer from 1to 5; and R₁₂ is an electrophilic or nucleophilic group for conjugatingthe acridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.

In some embodiments according to formula (I), G will be, at one or bothoccurrences, a group:

where R₂ and R₃ are independently at each occurrence a methyl group or agroup —(CH₂CH₂O)_(n)CH₃, where n is an integer from 1 to 5; and inparticular, G may be a group:

at one or both occurrences; or in another embodiment G may be a group:

at one or both occurrences. In a related embodiment, G is a group:

at one or both occurrences.

In other embodiments according to formula (I), G represents, at one orboth occurrences, a group:

where R₄, R₅, R₆ and R₇ are independently at each occurrence a methylgroup or a group —(CH₂CH₂O)_(n)CH₃, where n is an integer from 1 to 5.In one variant according to this embodiment, R₄-R₇ may represent methylgroups, such that G is a group:

at one or both occurrences.

In one embodiment according to formula (I), G is a group:

at one or both occurrences.

In the acridinium esters according to formula (I), R₁₂ may be selected,for example, from the group consisting of:

-   -   (1) —OH;    -   (2) —O—N-succinimidyl;    -   (3) —NH—(CH₂)₅—C(O)—O—N-succinimidyl;    -   (4) —NH—(CH₂)₅—COOH;    -   (5) —NH—(C₂H₄O)_(n)—C₂H₄NH—C(O)—(CH₂)₃—C(O)—O—N-succinimidyl        wherein n=1 to 5;    -   (6) —NH—(C₂H₄O)_(n)—C₂H₄NH—C(O)—(CH₂)₃—COOH, wherein n=1 to 5;    -   (7) —NH—(C₂H₄O)_(n)—C₂H₄NH₂, wherein n=1 to 5; and    -   (8) —NH—R—NHR, wherein R is independently hydrogen, alkyl,        alkenyl, alkynyl, or aralkyl; wherein R optionally comprises up        to 20 heteroatoms.

In various illustrative embodiments, R₁₂ will be —OH, or R₁₂ will be agroup:

—NH—(C₂H₄O)_(n)—C₂H₄NH₂, wherein n=1 to 5,

or R₁₂ will be a group:

—NH—(C₂H₄O)_(n)—C₂H₄NH—C(O)—(CH₂)₃—C(O)—O—R″, where n=1 to 5; and whereR″ is hydrogen or —N-succinimidyl.

One acridinium ester according to formula (I) has the followingstructure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.

Another acridinium ester according to formula (I) has the followingstructure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.

Yet another acridinium ester according to formula (I) has the followingstructure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.

Another acridinium ester according to formula (I) has the followingstructure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.

Still another acridinium ester according to formula (I) has thefollowing structure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.

In one exemplary embodiment of the acridinium esters according toformula (I), R₁₂ represents —OH.

In another aspect of the invention, an assay for the detection orquantification of an analyte is provided comprising the steps of: (a)providing a conjugate comprising: (i) a binding molecule specific for ananalyte; and (ii) a hydrophilic, high quantum yield and fast lightemitting acridinium ester according to formula (I); (b) providing asolid support having immobilized thereon a second binding moleculespecific for the analyte; (c) mixing the conjugate, the solid phase anda sample suspected of containing the analyte to form a binding complex;(d) separating the binding complex captured on the solid support; (e)triggering chemiluminescence of the binding complex from step (d) byadding chemiluminescence triggering reagents; (f) measuring the amountof light emission with a luminometer; and (g) detecting the presence orcalculating the concentration of the analyte by comparing the amount oflight emitted from the reaction mixture with a standard dose responsecurve which relates the amount of light emitted to a known concentrationof the analyte.

In a related aspect, an assay for the detection or quantification of ananalyte is provided comprising the steps of: (a) providing a conjugateof an analyte with a hydrophilic, high quantum yield and fast lightemitting acridinium ester according to formula (I); (b) providing asolid support immobilized with a binding molecule specific for theanalyte; (c) mixing the conjugate, solid support and a sample suspectedof containing the analyte to form a binding complex; (d) separating thebinding complex captured on the solid support; (e) triggering thechemiluminescence of the binding complex from step (d) by addingchemiluminescence triggering reagents; (f) measuring the amount of lightwith an luminometer; and (g) detecting the presence or calculating theconcentration of the analyte by comparing the amount of light emittedfrom the reaction mixture with a standard dose response curve whichrelates the amount of light emitted to a known concentration of theanalyte.

These and other aspects of the invention may be more clearly understoodby reference to the following detailed description of the invention andthe appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates structures of B-AEs with electrophilicN-hydroxysuccinimidyl (NHS) functional groups suitable for preparingconjugates of proteins or other molecules containing nucleophilicfunctional groups.

FIG. 2 illustrates B-AE structures with nucleophilic, hexaethyleneglycol amine (HEG-NH₂) functional groups useful for conjugating theacridinium compound to molecules containing electrophilic functionalgroups.

FIG. 3 shows the structures of estradiol conjugates (abbreviated asB-AE-E2) prepared using the B-AEs of FIG. 2.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The introduction of electron-donating functional groups such as OR* atC2 and/or C7 of the acridinium ring increases the quantum yield of thecorresponding chemiluminescent acridinium compound. When the R* group ishydrophilic, such as a sulfopropyl group or methoxy poly(ethylene)glycol, the corresponding acridinium compound not only exhibitsincreased light output but also shows reduced non-specific binding inimmunoassays. These two properties in conjunction lead to an increase inthe sensitivity of immunoassays.

The main objectives of the current invention were to identify structuralfeatures of acridinium compounds that result in (a) faster lightemission when compared to NSP-DMAE and derivatives as well as HQYAE; (b)improved stability especially when compared to HQYAE; (c) high lightoutput that is comparable to HQYAE and (d) low non-specific binding thatis comparable to HQYAE.

The hydrophilic acridinium compounds according to the present invention,abbreviated as B-AEs (Branched-Acridinium Esters), not only showincreased light output but also show improved stability and faster lightemission. By stability we refer to the chemiluminescent activity of theacridinium compounds. An increase in stability is thus manifested asincreased retention of chemiluminescent activity as a function of time.Increased stability of acridinium compounds is useful because reagentsderived from such compounds are less likely to show a deterioration ofassay performance as a function of time and moreover, the shelf life ofregents derived from such compounds is likely to be extended therebyleading to less waste. Typically, assay reagents derived from acridiniumcompounds include conjugates of proteins or small molecules. The secondproperty of the acridinium compounds is faster light emission by whichis meant that these compounds emit their total light in a significantlyshorter period of time compared to acridinium compounds lacking theunique structural features of the acridinium compounds of the currentinvention. Faster light emission enables faster measurements in assaysand has the potential to increase the throughput of automated analyzers.The throughput of automated analyzers is normally defined as the numberof tests the analyzer can perform in a given period of time. The thirdand fourth properties of the acridinium compounds of the currentinvention are their increased light output and low non-specific binding,both extremely useful for improving assay sensitivity.

It has unexpectedly been discovered that the placement of branchedfunctional groups derived from glycerol, of the type —OG, where G is abranched functional group, at C2 and/or C7 of the acridinium ringsignificantly increases the stability of the corresponding acridiniumcompound and leads to faster light emission. At the same time, thepresence of these branched functional groups increases the quantum yieldand lowers the non-specific binding of the corresponding acridiniumcompounds and their conjugates. Non-specific binding in assays usingsolid phases such as particles or microtiter plates are undesiredbinding interactions of conjugates to these solid phases. Theseundesired binding interactions typically increase the background of theassay leading to a net lowering of the signal to background ratio in theassay and thereby decreasing assay sensitivity.

The acridinium compounds of the current invention can be represented bythe general formula (I):

where R₁ is a methyl or sulfopropyl (—CH₂CH₂CH₂SO₃ ⁻) group; G isdefined as

where, R₂ and R₃ are the same or different and are —(CH₂CH₂O)_(n)Me,where n=1-5; R₄, R₅, R₆ and R₇ are the same or different and are eithera methyl group or —(CH₂CH₂O)_(n)Me, where n=1-5; and where R₁₂ is anelectrophilic or nucleophilic group.

More specifically, the acridinium compounds of the present invention canbe represented by the following formula:

where R₂ and R₃ are the same or different and are —(CH₂CH₂O)_(n)Megroups, where n=1-3; and where R₁₂ is selected from the group consistingof:

-   -   (1) —O—N-succinimidyl;    -   (2) —NH—(CH₂)₅—C(═O)—O—N-succinimidyl; and    -   (3) —NH—(C₂H₄O)_(n)—C₂H₄NH—C(═O)—(CH₂)₃—C(═O)—O—N-succinimidyl,        wherein n=1 to 5; and    -   (4) —NH—(C₂H₄O)_(n)—C₂H₄NH₂ where n=1-5.

The acridinium compounds of the current invention can also berepresented by the following formula:

where R₄, R₅, R₆ and R₇ are the same or different and are either methylor —(CH₂CH₂O)_(n)Me, where n=1-3; and where R₁₂ is selected from thegroup consisting of:

-   -   (1) —O—N-succinimidyl (NHS);    -   (2) —NH—(CH₂)₅—C(═O)—O—N-succinimidyl; and    -   (3) —NH—(C₂H₄O)_(n)—C₂H₄NH—C(═O)—(CH₂)₃—C(═O)—O—N-succinimidyl,        wherein n=1 to 5; and    -   (4) —NH—(C₂H₄O)_(n)—C₂H₄NH₂ where n=1-5.

Representative examples of the above general structures were synthesizedas discrete structures using traditional organic chemistry techniques.The structures of these compounds along with their abbreviatednomenclature are illustrated in FIGS. 1 and 2. FIG. 1 illustratesstructures of B-AEs with electrophilic N-hydroxysuccinimidyl (NHS)functional groups whereas FIG. 2 illustrates B-AE structures withnucleophilic, hexaethylene glycol amine (HEG-NH₂) functional groups. Theformer compounds are suitable for preparing conjugates of proteins orother molecules containing nucleophilic functional groups. The lattercompounds are also useful for conjugating the acridinium compound tomolecules containing electrophilic functional groups. FIG. 3 shows thestructures of estradiol conjugates (abbreviated as B-AE-E2) preparedusing the B-AEs of FIG. 2. Estradiol is a steroidal hormone that iscommonly measured by immunoassay.

The B-AEs of FIG. 1 as well as NSP-DMAE, NSP-DMAE-HEG-glutarate-NHS(abbreviated as HEG-AE) and the high quantum yield acridinium esterNSP-2,7-(OMHEG)2-DMAE-AC-NHS (abbreviated as HQYAE) were used to prepareconjugates of a murine, monoclonal anti-TSH antibody (TSH=thyroidstimulating hormone) as described in Example 9. Light emission from eachconjugate was triggered by the addition of two reagents. The firstreagent comprised 0.5% hydrogen peroxide in 100 mM nitric acid while thesecond reagent contained a surfactant in 0.25 N sodium hydroxide. Lightwas measured using a luminometer equipped with a photo-multiplier tubeas the detector. The amount of light emitted by each acridinium compoundconjugate was reported as Relative Light Units (RLUs) by theluminometer. The total amount of light emitted (100% RLUs) was measuredfor the various conjugates and light emission at shorter measurementtimes, are represented as fractions of this number and are alsoexpressed as percentages in Table 1. Other details pertaining to thesemeasurements can be found in the Examples section.

TABLE 1 % RLU as a function of measurement time of acridiniumcompound-anti-TSH antibody conjugates. Entry Conjugate 0.5 s 1.0 s 2.0 s5.0 s 1 HEG-AE 32 65 87 97 2 HQYAE 31 61 82 95 3 B1-AE 59 91 96 97 4B2-AE 62 91 96 98 5 B3-AE 57 89 96 97 6 B04-AE 90 99 99 99 7 B4-AE 68 9598 99

From Table 1, while HEG-AE and HQYAE emit only 65% and 61% of theirtotal light in one second, all the B-AE conjugates show much fasterlight emission with ≧89% of the light emitted in one second. The uniquestructural features in the B-AEs thus speed up light emission from thesecompounds when conjugated to a protein.

Similarly, the kinetics of light emission from the B-AE-E2 conjugatesillustrated in FIG. 3 was compared with light emission from the E2conjugates of NSP-DMAE-E2 and HQYAE-E2. Both the latter compoundsincorporated the same HEG linkers. The results of these measurements aretabulated in Table 2.

TABLE 2 % RLU as a function of measurement time of E2 conjugates EntryConjugate 0.5 s 1.0 s 2.0 s 5.0 s 1 NSP-DMAE 8 23 45 80 2 HQYAE 35 72 9198 3 B1-AE 33 71 93 98 4 B2-AE 33 70 92 97 5 B4-AE 39 77 95 99

For the estradiol conjugates, all the B-AEs again show faster lightemission when compared to NSP-DMAE-HEG-E2 conjugate.

In addition to showing fast light emission, the acridinium esters of thepresent invention also show good stability. By “stability,” is meant aminimal loss of chemiluminescent activity as measured by the loss ofRLUs when the compounds or conjugates are stored in an aqueous solutiontypically, in the pH range of 7-8, which is within the physiological pH.From a mechanistic viewpoint, hydrolysis of the phenolic ester is themain pathway by which chemiluminescent acridinium esters becomenon-chemiluminescent. Stable conjugates ensure long shelf life foracridinium ester reagents and also ensure that assay performance doesnot vary greatly over a given period of time. The stability of variousacridinium ester conjugates of the current invention are listed inTables 3 and 4. Aqueous solutions of the conjugates were stored at 37°C. in an aqueous buffer at pH 7.7 and RLUs were recorded periodicallyusing a luminometer. The RLUs that were measured at the initial timepoint, also referred to as day 0, were assigned a value of 100%. TheRLUs that were measured at other time points, are expressed aspercentages of this number. Other details pertaining to thesemeasurements can be found in the Examples section.

TABLE 3 Stability of anti-TSH antibody conjugates expressed as % RLUTime HEG- (days) AE HQYAE B1-AE B2-AE B3-AE B4-AE 0 100 100 100 100 100100 7 94 86 97 91 92 96 16 89 77 91 85 87 93 23 82 68 84 78 81 85 33 8268 87 79 83 86

TABLE 4 Stability of E2 conjugates expressed as % RLU Time HEG- (days)AE HQYAE B1-AE B2-AE B4-AE 0 100 100 100 100 100 1 99 98 97 98 98 5 9589 94 94 96 8 97 87 92 93 96 12 94 76 89 88 92 20 92 73 84 85 88 27 8966 77 81 84 33 84 60 75 77 82

As is evident from Tables 3 and 4, the B-AE conjugates retain a greaterproportion of their chemiluminescent activity and are more stablecompared to the HQYAE conjugate. For example, the anti-TSH antibodyconjugate of HQYAE retains 68% of its chemiluminescent activity after 33days at 37° C., the B-AE conjugates retain ≧79% of theirchemiluminescent activity in the same period of time. A similar trend isnoted for the estradiol (E2) conjugates where the B-AE conjugates retain≧75% of their chemiluminescent activity after 33 days at 37° C., whereasthe HQYAE conjugate's chemiluminescent activity has dropped to 60% inthe same time period.

In addition to showing fast light emission, the B-AEs of the presentinvention also show increased light output that is comparable to orbetter than HQYAE. Table 5 summarizes the relative quantum yields of thevarious B-AEs when conjugated to the anti-TSH monoclonal antibody. Inthis table, the quantum yield of HEG-AE was assigned a value of unity(1) and the quantum yields of all the other conjugates are relative tothis conjugate of this compound.

TABLE 5 Relative quantum yields of AE conjugates of anti-TSH antibodyRelative quantum Entry Conjugate yield 1 HEG-AE 1.0 2 HQYAE 2.2 3 B1-AE4.7 4 B2-AE 2.7 5 B3-AE 1.9 6 B04-AE 1.5 7 B4-AE 2.0

As can be noted from Table 5, all the B-AE conjugates show greater lightoutput (higher quantum yield) than the HEG-AE conjugate and are eithercomparable or greater than the light output of the HQYAE conjugate.

Finally, the B-AEs of the current invention also show low non-specificbinding to solid phases (Table 6). Non-specific binding, as describedearlier, in assays using solid phases such as particles or microtiterplates are undesired binding interactions of conjugates to these solidphases. These undesired binding interactions typically increase thebackground of the assay leading to a net lowering of the signal tobackground ratio in the assay and thereby decreasing assay sensitivity.For the various acridinium conjugates of the anti-TSH antibody listed inTable 6, non-specific binding was measured on two different kinds ofparticles; paramagnetic particles (PMP) and magnetic latex particles(MLP). These two particles differ in their intrinsic composition. PMPsare made mainly of iron oxide particles with a silane coating containingamines. The amines are used to cross-link proteins to the particlesurface using reagents such as glutaraldehyde. MLPs on the other handare made of polystyrene. The MLPs used in Table 6 contained a thin layerof magnetite to enable magnetic separation and a polyacrylic acidcoating for conjugating proteins. The two types of particles were mixedwith solutions of the conjugates for a specific period of time and thenthe particles were magnetically separated, washed once and then thechemiluminescence associated with the particles was measured.(Experimental details can be found in Example 11.) The ratio of thischemiluminescence value in comparison to the total chemiluminescenceinput is referred to fraction non-specific binding (fNSB). Conjugateswith low non-specific binding will have low fNSB values. In examiningTable 6, it is evident that all the B-AE conjugates have lowernon-specific binding than HEG-AE on both types of particles. The fNSBvalues of the B-AE conjugates were also found to be comparable to thepreviously described hydrophilic HQYAE.

TABLE 6 Fractional Nonspecific Binding (fNSB) of anti-TSHantibody-acridinium conjugates to particles. Particle PMP MLP AcridiniumEster Conjugate Fractional Nonspecific Binding (fNSB) HEG-AE 4.1E−051.2E−05 HQYAE 6.0E−06 7.5E−07 B1-AE 4.5E−06 1.8E−06 B2-AE 5.9E−062.8E−06 B3-AE 3.9E−06 1.2E−06 B4-AE 5.8E−06 1.9E−06 B04-AE 5.9E−067.1E−06

The hydrolytically stable, fast light emitting, hydrophilic, highquantum yield acridinium compounds of the invention are useful as labelsin assays for the determination or quantitation of analytes. Analytesthat are typically measured in such assays are often substances of someclinical relevance and can span a wide range of molecules from largemacromolecules such as proteins, nucleic acids, viruses bacteria, etc.to small molecules such as ethanol, vitamins, steroids, hormones,therapeutic drugs, etc. A ‘sandwich’ immunoassay typically involves thedetection of a large molecule, also referred to as macromolecularanalyte, using two binding molecules such as antibodies. One antibody isimmobilized or attached to a solid phase such as a particle, bead,membrane, microtiter plate or any other solid surface. Methods for theattachment of binding molecules such as antibodies to solid phases arewell known in the art. For example, an antibody can be covalentlyattached to a particle containing amines on its surface by using across-linking molecule such as glutaraldehyde. The attachment may alsobe non-covalent and may involve simple adsorption of the bindingmolecule to the surface of the solid phase, such as polystyrene beadsand microtiter plate. The second antibody is often covalently attachedwith a chemiluminescent or fluorescent molecule often referred to as alabel. Labeling of binding molecules such as antibodies and otherbinding proteins are also well known in the art and are commonly calledconjugation reactions and the labeled antibody is often called aconjugate. Typically, an amine-reactive moiety on the label reacts withan amine on the antibody to form an amide linkage. Other linkages suchas thioether, ester, carbamate, and the like, between the antibody andthe label are also well known. In the assay, the two antibodies bind todifferent regions of the macromolecular analyte. The macromolecularanalyte can be, for example, proteins, nucleic acids, oligosaccharides,antibodies, antibody fragments, cells, viruses, receptors, or syntheticpolymers. The binding molecules can be antibodies, antibody fragments,nucleic acids, peptides, binding proteins or synthetic binding polymers.For example the folate binding protein (“FBP”) binds the analyte folate.Synthetic binding molecules that can bind a variety of analytes havealso been disclosed by Mossbach et al. Biotechnology vol. 14, pp.163-170 (1995).

When the solid phase with the immobilized antibody and the labeledantibody is mixed with a sample containing the analyte, a bindingcomplex is formed between the analyte and the two antibodies. This typeof assay is often called a heterogenous assay because of the involvementof a solid phase. The chemiluminescent or fluorescent signal associatedwith the binding complex can then be measured and the presence orabsence of the analyte can be inferred. Usually, the binding complex isseparated from the rest of the binding reaction components such asexcess, labeled antibody prior to signal generation. For example if thebinding complex is associated with a magnetic bead, a magnet can be usedto separate the binding complex associated with the bead from bulksolution. By using a series of ‘standards’, that is, knownconcentrations of the analyte, a ‘dose-response’ curve can be generatedusing the two antibodies. Thus, the dose-response curve correlates acertain amount of measured signal with a specific concentration ofanalyte. In a sandwich assay, as the concentration of the analyteincreases, the amount of signal also increases. The concentration of theanalyte in an unknown sample can then be calculated by comparing thesignal generated by an unknown sample containing the macromolecularanalyte, with the dose-response curve.

In a similar vein, the two binding components can also be nucleic acidsthat bind or hybridize to different regions of a nucleic acid analyte.The concentration of the nucleic acid analyte can then be deduced in asimilar manner.

Another class of immunoassays for small molecule analytes such assteroids, vitamins, hormones, therapeutic drugs or small peptidesemploys an assay format that is commonly referred to as a competitiveassay. Typically, in a competitive assay, a conjugate is made of theanalyte of interest and a chemiluminescent or fluorescent label bycovalently linking the two molecules. The small molecule analyte can beused as such or its structure can be altered prior to conjugation to thelabel. The analyte with the altered structure is called an analog. It isoften necessary to use a structural analog of the analyte to permit thechemistry for linking the label with the analyte. Sometimes a structuralanalog of an analyte is used to attenuate or enhance its binding to abinding molecule such an antibody. Such techniques are well known in theart. The antibody or a binding protein to the analyte of interest isoften immobilized on a solid phase either directly or through asecondary binding interaction such as the biotin-avidin system.

The concentration of the analyte in a sample can be deduced in acompetitive assay by allowing the analyte-containing sample and theanalyte-label conjugate to compete for a limited amount of solidphase-immobilized binding molecule. As the concentration of analyte in asample increases, the amount of analyte-label conjugate captured by thebinding molecule on the solid phase decreases. By employing a series of‘standards’, that is, known concentrations of the analyte, adose-response curve can be constructed where the signal from theanalyte-label conjugate captured by the binding molecule on the solidphase is inversely correlated with the concentration of analyte. Once adose-response curve has been devised in this manner, the concentrationof the same analyte in an unknown sample can be deduced by comparing thesignal obtained from the unknown sample with the signal in thedose-response curve.

Another format of the competitive assay for small molecules analytesinvolves the use of a solid phase that is immobilized with the analyteof interest or an analyte analog and an antibody or a binding proteinspecific for the analyte that is conjugated with a chemiluminescent orfluorescent label. In this format, the antibody-label conjugate iscaptured onto the solid phase through the binding interaction with theanalyte or the analyte analog on the solid phase. The analyte ofinterest present in a sample then “competitively” binds to theantibody-label conjugate and thus inhibits or replaces the interactionof the antibody-label conjugate with the solid phase. In this fashion,the amount of signal generated from the antibody-label conjugatecaptured on the solid phase is correlated to the amount of the analytein sample.

In accordance with the foregoing, an assay for the detection orquantification of an analyte comprises, according to one embodiment ofthe invention, the following steps:

(a) providing a conjugate comprising: (i) a binding molecule specificfor an analyte; and (ii) any of the inventive hydrophilic, high quantumyield and fast light emitting acridinium esters according to theinvention;

(b) providing a solid support having immobilized thereon a secondbinding molecule specific for said analyte;

(c) mixing the conjugate, the solid phase and a sample suspected ofcontaining the analyte to form a binding complex;

(d) separating the binding complex captured on the solid support;

(e) triggering chemiluminescence of the binding complex from step (d) byadding chemiluminescence triggering reagents;

(f) measuring the amount of light emission with a luminometer; and

(g) detecting the presence or calculating the concentration of theanalyte by comparing the amount of light emitted from the reactionmixture with a standard dose response curve which relates the amount oflight emitted to a known concentration of the analyte.

In another embodiment, an assay for the detection or quantification ofan analyte is provided comprising the steps of:

(a) providing a conjugate of an analyte with any of the any of theinventive hydrophilic, high quantum yield and fast light emittingacridinium esters (b) providing a solid support immobilized with abinding molecule specific for the analyte;

(c) mixing the conjugate, solid support and a sample suspected ofcontaining the analyte to form a binding complex;

(d) separating the binding complex captured on the solid support;

(e) triggering the chemiluminescence of the binding complex from step(d) by adding chemiluminescence triggering reagents;

(f) measuring the amount of light with an luminometer; and

(g) detecting the presence or calculating the concentration of theanalyte by comparing the amount of light emitted from the reactionmixture with a standard dose response curve which relates the amount oflight emitted to a known concentration of the analyte.

Macromolecular analytes can be proteins, nucleic acids,oligosaccharides, antibodies, antibody fragments, cells, viruses,synthetic polymers, and the like. Small molecule analytes can besteroids, vitamins, hormones, therapeutic drugs, small peptides, and thelike. The binding molecules in the assays can be an antibody, anantibody fragment, a binding protein, a nucleic acid, a peptide, areceptor or a synthetic binding molecule.

EXAMPLE 1 Synthesis of B1-AE-NHS ester, 1i

a) 1,3-Bis(methoxyethoxy)-2-propyl toluenesulfonate, 1b. The compound1a, 1,3-bis(methoxyethoxy)-2-propanol was synthesized as described byCormier and Gregg in Chem. Mater. 1998, 10, 1309-1319. A solution of 1a(2 g, 9.6 mmol) in anhydrous pyridine (10 mL) was treated with4-dimethylaminopyridine (0.234 g, 1.92 mmol) followed byp-toluenesulfonyl chloride (3.67 g, 19.25 mmol). The reaction wasstirred at room temperature under a nitrogen atmosphere for 3 days. Thesolvent was then removed under reduced pressure and the residue waspartitioned between ethyl acetate (75 mL) and 10% HCl (100 mL). Theethyl acetate layer was washed with brine, dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. The crudeproduct (4.05 g) was purified by flash chromatography on silica gelusing 1:1, ethyl acetate/hexanes as eluent. The product was recovered asa light yellow oil. Yield=2.42 g (70%).

b) Compound 1d. A mixture of 2,7-dihydroxy acridine methyl ester, 1c(0.2 g, 0.48 mmol), (U.S. Pat. No. 7,309,615), compound 1b (0.868 g,2.39 mmol) and cesium carbonate (0.39 g, 1.2 mmol) in anhydrous DMF (10mL) was heated at 100° C. under a nitrogen atmosphere for 4-5 hours. Asmall portion of the reaction mixture was then analyzed by HPLC using aPhenomenex, C₁₈ 4.6 mm×25 cm column and a 30 minute gradient of 10→100%B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at a flow rate of 1.0mL/minute and UV detection at 260 nm. Product was observed eluting atRt=25 minutes and was the major component. The reaction was then cooledto room temperature and concentrated under reduced pressure. The residuewas partitioned between ethyl acetate (50 mL) and water (50 mL). Theethyl acetate layer was separated, dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. The crude product (0.48g) was purified by flash chromatography on silica gel using ethylacetate as eluent. Yield=0.134 g (34%); MALDI-TOF MS 797.8 observed.

c) Compound 1f. A mixture of compound 1d (60 mg, 75.3 umoles), distilled1,3-propane sultone (1 g, 8.2 mmol) and sodium bicarbonate (65 mg, 0.77mmol) was heated at 150° C. under a nitrogen atmosphere for 1 hour. Aportion of the reaction mixture was withdrawn, diluted with methanol andanalyzed by HPLC as described in section (b). The acridinium ester 1ewas observed eluting at Rt=19 minutes. The reaction was cooled to roomtemperature and 20 mL of 1:1, ethyl acetate/hexanes was added. Themixture was sonicated briefly to disperse the gummy solid and thesolvent was then decanted. The crude product was dried under reducedpressure. This crude product was suspended in 1 N HCl (10 mL) and wasrefluxed under a nitrogen atmosphere for 2 hours. HPLC analysis of thecrude reaction mixture showed complete hydrolysis of the reactionmixture with product eluting at Rt=16 minutes. The product was purifiedby preparative HPLC using an YMC, C₁₈ 30×300 mm column and the samegradient at described in section (b) at a solvent flow rate of 20mL/minute and UV detection at 260 nm. The HPLC fractions containingproduct were combined and concentrated under reduced pressure. Yield=55mg (81%); MALDI-TOF MS 904.7 observed.

d) Compound 1g. A solution of compound 1f (53 mg, 58.2 umoles) inanhydrous DMF (2 mL) was treated with diisopropylethylamine (15.2 uL,87.3 umoles) and TSTU (20 mg, 64 umoles). The reaction was stirred atroom temperature. After 30 minutes, HPLC analysis of the reactionmixture as described in section (b) indicated complete conversion to theNHS ester eluting at Rt=18 minutes. This reaction was added dropwise toa stirred solution of 2,2′-(ethylenedioxy)bis(ethylamine) (86 ul, 0.582mmol) in anhydrous DMF (1.0 mL). After 30 minutes, HPLC analysis of thereaction mixture, as described in section (b) showed complete conversionto the product 1g eluting at Rt=13.6 minutes. The product was purifiedby preparative HPLC as described in section (c). Yield=50 mg (83%);MALDI-TOF MS 1035.6 observed.

e) B1-AE-NHS, compound 1i. A solution of compound 1g (47.5 mg, 46umoles) in anhydrous methanol (3 mL) was treated withdiisopropylethylamine (40 uL, 0.23 mmol) and glutaric anhydride (26 mg,0.23 mmol). The reaction was stirred at room temperature. After 30minutes, HPLC analysis, as described in section (b), showed completeconversion to the glutarate derivative 1h eluting at Rt=14.8 minutes.The reaction mixture was diluted with anhydrous toluene (3 mL) andconcentrated under reduced pressure. The crude product was dissolved inanhydrous DMF (3 mL) and treated with diisopropylethylamine (80 uL, 0.46mmol) and TSTU (138 mg, 0.46 mmol). After stirring for 30 minutes, HPLCanalysis, as described in section (b), showed >80% conversion to theproduct 1i eluting at Rt=16 minutes. The product was purified bypreparative HPLC as described in section (c). The HPLC fractionscontaining product were frozen at −80° C. and lyophilized to dryness.The lyophilized product was dissolved in anhydrous MeCN and transferredto a tared round bottom flask and concentrated under reduced pressure.Yield=32 mg (56%); MALDI-TOF MS 1247.1 observed.

The following reactions describe the synthesis of B1-AE-NHS, compound1i.

EXAMPLE 2 Synthesis of B2-AE-NHS ester, 2h

a). 1,3-Bis(3,6-dioxaheptanyl)glycerol-2-toluenesulfonate, 2b. Thecompound 1,3-Bis(3,6-dioxaheptanyl)glycerol, 2a, was synthesized asdescribed by Vacus and Simon in Adv. Mater. 1995, 7, 797-800. Crude 2a(16 g, 0.054 mol) was dissolved in anhydrous pyridine (50 mL) andtreated with 4-dimethylaminopyridine (1.32 g, 0.011 mol) followed byp-toluenesulfonyl chloride (12.4 g, 0.065 mol). The reaction was stirredunder a nitrogen atmosphere for 16 hours. The solvent was then removedunder reduced pressure and the residue was partitioned between ethylacetate (100 mL) and 2N HCl (100 mL). The ethyl acetate layer was washedwith saturated sodium bicarbonate solution followed by brine. It wasthen dried over magnesium sulfate and concentrated under reducedpressure. The crude product (14 g) was purified by flash chromatographyon silica gel using 1:4, hexanes/ethyl acetate as eluent. Yield=6.3 g,light yellow oil. MALDI-TOF MS 473.4 observed, (M+Na⁺).

b) Compound 2c. A mixture of 1c (0.2 g, 0.48 mmol), 2b (1.08 g, 2.4mmol) and cesium carbonate (0.39 g, 0.12 mmol) in anhydrous DMF (10 mL)was heated in an oil bath at 100° C. under a nitrogen atmosphere. After5 hours, the reaction was cooled to room temperature and concentratedunder reduced pressure. The residue was partitioned between ethylacetate (75 mL) and water (75 mL). The ethyl acetate layer was washedwith brine and dried over anhydrous magnesium sulfate. The solvent wasthen removed under reduced pressure to afford 0.974 g of crude productwhich was purified by flash chromatography on silca gel using 3%methanol in ethyl acetate as eluent. Yield=99.4 mg (21%); MALDI-TOF MS974.4 observed.

c) Compound 2e. A mixture of compound 2c (58 mg, 60 umoles), distilled1,3-propane sultone (0.75 g, 6.15 mmol) and sodium bicarbonate (50 mg,0.59 mmol) was heated at 150° C. under a nitrogen atmosphere. After 1hour, a small portion was withdrawn, diluted with methanol and analyzedby HPLC using a Phenomenex, C₁₈ 4.6 mm×25 cm column and a 30 minutegradient of 10→100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) ata flow rate of 1.0 mL/minute and UV detection at 260 nm. Product wasobserved eluting at Rt=18.5 minutes (>80% conversion, startimg materialRt=23.5 minutes). The reaction was cooled to room temperature and 20 mLof 1:1, ethyl acetate/hexanes was added. After brief sonication todisperse the gummy product, the solvent was decanted and the product 2dwas dried under vacuum.

The crude acridinium ester 2d was suspended in I N HCl (10 mL) andrefluxed under a nitrogen atmosphere for 2 hours. HPLC analysis, asdescribed above, indicated complete conversion to product 2e eluting at16 minutes. The product was purified by preparative HPLC using an YMC,C₁₈ 30×300 mm column and the same gradient described above at a solventflow rate of 20 mL/minute and UV detection at 260 nm. The HPLC fractionscontaining product were combined and concentrated under reducedpressure. Yield=42 mg (65%); MALDI-TOF MS 1083.3 observed.

d) Compound 2f. A solution of compound 2e (42 mg, 39 umoles) inanhydrous DMF (2 mL) was treated with diisopropylethylamine (10 uL, 59umoles) and TSTU (14 mg, 46.5 umoles). The reaction was stirred at roomtemperature. After 15 minutes, HPLC analysis, as described in section(c) showed complete conversion to the NHS ester eluting at Rt=17.7minutes. This reaction was added dropwise to a stirred solution of2,2′-(ethylenedioxy)bis(ethylamine) (58 ul, 0.39 mmol) in anhydrous DMF(1.0 mL). After 30 minutes, HPLC analysis of the reaction mixture, asdescribed in section (b) showed complete conversion to the product 2feluting at Rt=13.6 minutes. The product was purified by preparative HPLCas described in section (c). Yield=37 mg (79%); MALDI-TOF MS 1217.9observed.

e) B2-AE-NHS, compound 2h. A solution of compound 2f (37 mg, 30 umoles)in anhydrous methanol (3 mL) was treated with diisopropylethylamine (26uL, 0.15 mmol) and glutaric anhydride (17 mg, 0.15 mmol). The reactionwas stirred at room temperature. After 30 minutes, HPLC analysis, asdescribed in section (c), showed complete conversion to the glutaratederivative 2g eluting at Rt=15 minutes. The reaction mixture was dilutedwith anhydrous toluene (3 mL) and concentrated under reduced pressure.The crude product was dissolved in anhydrous DMF (2 mL) and treated withdiisopropylethylamine (52 uL, 0.3 mmol) and TSTU (89 mg, 0.3 mmol).After stirring for 30 minutes, HPLC analysis, as described in section(c), showed >80% conversion to the product 2h eluting at Rt=16 minutes.The product was purified by preparative HPLC as described in section(c). The HPLC fractions containing product were frozen at −80° C. andlyophilized to dryness. The lyophilized product was dissolved inanhydrous MeCN and transferred to a tared round bottom flask andconcentrated under reduced pressure. Yield=39 mg (91%); MALDI-TOF MS1425.4 observed.

The following reactions describe the synthesis of B2-AE-NHS, 2h.

EXAMPLE 3 B3-AE-NHS ester 3h

a) 1,3-Bis(3,6,9-dioxadecanyl)glycerol-2-toluenesulfonate, 3b. Thecompound 1,3-Bis(3,6,9-dioxadecanyl)glycerol, 3a, was synthesized asdescribed by Lauter et al. in Macromol. Chem. Phys. 1998, 199,2129-2140. The alcohol (7 g, 0.0182 mol) was dissolved in anhydrouspyridine (30 mL) and treated with 4-dimethylaminopyridine (0.444 g, 3.6mmol) and p-toluenesulfonyl chloride (3.85 g, 0.02 mol). The reactionwas stirred under a nitrogen atmosphere for 3 days. The solvent was thenremoved under reduced pressure and the residue was partitioned betweenethyl acetate (100 mL) and 10% HCl (100 mL). The ethyl acetate layer waswashed with saturated sodium bicarbonate solution and brine. It was thendried over anhydrous magnesium sulfate and concentrated under reducedpressure. The crude product was purified by flash chromatography onsilica gel using 5:4.5:0.5, hexanes:ethyl acetate:methanol. Yield=4.47 g(45%); light yellow oil.

b) Compound 3c. A mixture of 1c (0.2 g, 0.48 mmol), 3b (1.3 g, 2.4 mmol)and cesium carbonate (0.35 g, 0.11 mmol) in anhydrous DMF (10 mL) washeated in an oil bath at 100° C. under a nitrogen atmosphere. After 6hours, the reaction was cooled to room temperature and concentratedunder reduced pressure. The residue was partitioned between ethylacetate (75 mL) and water (75 mL). The ethyl acetate layer was washedwith brine and dried over anhydrous magnesium sulfate. The solvent wasthen removed under reduced pressure to afford 1.3 g of crude productwhich was purified by flash chromatography on silca gel using 5%methanol in ethyl acetate as eluent. Yield=134 mg (22%); MALDI-TOF MS1148.9 observed.

c) Compound 3e. A mixture of compound 3c (45 mg, 39 umoles), distilled1,3-propane sultone (0.5 g, 4.1 mmol) and sodium bicarbonate (33 mg,0.39 mmol) was heated at 150° C. under a nitrogen atmosphere. After 2hours, a small portion was withdrawn, diluted with methanol and analyzedby HPLC using a Phenomenex, C₁₈ 4.6 mm×25 cm column and a 30 minutegradient of 10→100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) ata flow rate of 1.0 mL/minute and UV detection at 260 nm. Product wasobserved eluting at Rt=18.3 minutes (>80% conversion, startimg materialRt=22.5 minutes). The reaction was cooled to room temperature and 20 mLof 1:1, ethyl acetate/hexanes was added. After brief sonication todisperse the gummy product, the solvent was decanted and the product 3dwas dried under vacuum.

The crude acridinium ester 3d was suspended in I N HCl (10 mL) andrefluxed under a nitrogen atmosphere for 2 hours. HPLC analysis, asdescribed above, indicated complete conversion to product 3e eluting at16.3 minutes. The product was purified by preparative HPLC using an YMC,C₁₈ 30×300 mm column and the same gradient at described above at asolvent flow rate of 20 mL/minute and UV detection at 260 nm. The HPLCfractions containing product were combined and concentrated underreduced pressure. Yield=28 mg (57%); MALDI-TOF MS 1255.9 observed.

d) Compound 3f A solution of compound 3e (28 mg, 22.3 umoles) inanhydrous DMF (2 mL) was treated with diisopropylethylamine (6.4 uL,33.5 umoles) and TSTU (8 mg, 26.6 umoles). The reaction was stirred atroom temperature. After 30 minutes, HPLC analysis, as described insection (c) showed complete conversion to the NHS ester eluting atRt=17.7 minutes. This reaction was added dropwise to a stirred solutionof 2,2′-(ethylenedioxy)bis(ethylamine) (32 ul, 0.22 mmol) in anhydrousDMF (1.0 mL). After one hour, HPLC analysis of the reaction mixture, asdescribed in section (c) showed complete conversion to the product 3feluting at Rt=14 minutes. The product was purified by preparative HPLCas described in section (c). Yield=28 mg (90%); MALDI-TOF MS 1388.6observed.

e) B3-AE-NHS, compound 3h. A solution of compound 3f (28 mg, 20 umoles)in anhydrous methanol (2 mL) was treated with diisopropylethylamine(17.6 uL, 0.1 mmol) and glutaric anhydride (11.5 mg, 0.1 mmol). Thereaction was stirred at room temperature. After 30 minutes, HPLCanalysis, as described in section (c), showed complete conversion to theglutarate derivative 3g eluting at Rt=15.2 minutes. The reaction mixturewas diluted with anhydrous toluene (3 mL) and concentrated under reducedpressure. The crude product was dissolved in anhydrous DMF (2 mL) andtreated with diisopropylethylamine (35 uL, 0.2 mmol) and TSTU (60 mg,0.2 mmol). After stirring for 30 minutes, HPLC analysis, as described insection (c), showed >70% conversion to the product 3h eluting at Rt=16.2minutes. The product was purified by preparative HPLC as described insection (c). The HPLC fractions containing product were frozen at −80°C. and lyophilized to dryness. The lyophilized product was dissolved inanhydrous MeCN and transferred to a tared round bottom flask andconcentrated under reduced pressure. Yield=17.6 mg (55%); MALDI-TOF MS1598 observed.

The following reactions describe the synthesis of B3-AE-NHS, 3h.

EXAMPLE 4 B4-AE-NHS ester, 4h

a) Compound 4a. 1,3-Bis(methoxyethoxy)-2-propanol, 1a, (12 g, 0.058 mol)and potassium hydroxide (2.43 g, 0.043 mol) were stirred vigorously andepichlorohydrin (1.334 g, 0.0144 mol) was added dropwise. The reactionwas heated at 80° C. for 24 hours. It was then cooled to roomtemperature and water (50 mL) was added. The solution was extracted withdichloromethane (3×50 mL). The combined dichloromethane extracts weredried over anhydrous magnesium sulfate and concentrated under reducedpressure. The crude product (10.85 g) was sued as such for the nextreaction.

b) Compound 4b. A solution of 4a (10.5 g crude, 0.023 mol) in anhydrouspyridine (25 mL) was treated with 4-dimethylaminopyridine (0.56 g, 4.6mmol) and p-toluenesulfonyl chloride (0.046 mol, 8.8 g). The reactionwas stirred at room temperature under a nitrogen atmosphere for 16hours. The solvent was then removed under reduced pressure and theresidue was partitioned between ethyl acetate (100 mL) and 2 N HCl (100mL). The ethyl acetate layer was washed with saturated sodiumbicarbonate and brine. It was then dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. The crude product (16.4g) was purified by flash chromatography on silica gel using 1:1 ethylacetate:hexanes to elute 1,3-bis(methoxyethoxy)-2-propyltoluenesulfonate followed by ethyl acetate to elute product. Yield=2.82g (32%); MALDI-TOF MS 648.6 (M+Na⁺).

c) Compound 4c. A mixture of 2,7-dihydroxy acridine methyl ester, 1c(0.2 g, 0.48 mmol) compound 4b (1.5 g, 2.4 mmol) and cesium carbonate(0.39 g, 1.2 mmol) in anhydrous DMF (10 mL) was heated at 100° C. undera nitrogen atmosphere for 4-5 hours. A small portion of the reactionmixture was then analyzed by HPLC using a Phenomenex, C₁₈ 4.6 mm×25 cmcolumn and a 30 minute gradient of 10→100% B (A=water with 0.05% TFA,B=MeCN with 0.05% TFA) at a flow rate of 1.0 mL/minute and UV detectionat 260 nm. Product was observed eluting at Rt=23.5 minutes and was themajor component. The reaction was then cooled to room temperature andconcentrated under reduced pressure. The residue was partitioned betweenethyl acetate (50 mL) and water (50 mL). The ethyl acetate layer wasseparated, dried over anhydrous magnesium sulfate and concentrated underreduced pressure. The crude product (1.36 g) was purified by flashchromatography on silica gel using 5% methanol in ethyl acetate aseluent. Yield=0.156 g (25%); MALDI-TOF MS 1325 observed.

d) Compound 4e. A mixture of compound 4c (60 mg, 45.3 umoles), distilled1,3-propane sultone (1.0 g, 8.2 mmol) and sodium bicarbonate (76 mg, 0.9mmol) was heated at 150° C. under a nitrogen atmosphere. After 2 hours,a small portion was withdrawn, diluted with methanol and analyzed byHPLC using a Phenomenex, C₁₈ 4.6 mm×25 cm column and a 30 minutegradient of 10→100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) ata flow rate of 1.0 mL/minute and UV detection at 260 nm. Product wasobserved eluting at Rt=17.8 minutes (>80% conversion, startimg materialRt=23.5 minutes). The reaction was cooled to room temperature and 20 mLof 1:1, ethyl acetate/hexanes was added. After brief sonication todisperse the gummy product, the solvent was decanted and the product 4dwas dried under vacuum.

The crude acridinium ester 4d was suspended in I N HCl (10 mL) andrefluxed under a nitrogen atmosphere for 2 hours. HPLC analysis, asdescribed above, indicated complete conversion to product 4e eluting at17 minutes. The product was purified by preparative HPLC using an YMC,C₁₈ 30×300 mm column and the same gradient at described above at asolvent flow rate of 20 mL/minute and UV detection at 260 nm. The HPLCfractions containing product were combined and concentrated underreduced pressure. Yield=33.5 mg (52%); MALDI-TOF MS 1433.1 observed.

e) Compound 4f A solution of compound 4e (33.5 mg, 23.4 umoles) inanhydrous DMF (2 mL) was treated with diisopropylethylamine (6.1 uL, 35umoles) and TSTU (8.5 mg, 28.2 umoles). The reaction was stirred at roomtemperature. After 30 minutes, HPLC analysis, as described in section(c) showed complete conversion to the NHS ester eluting at Rt=18.7minutes. This reaction was added dropwise to a stirred solution of2,2′-(ethylenedioxy)bis(ethylamine) (35 ul, 0.24 mmol) in anhydrous DMF(1.0 mL). After one hour, HPLC analysis of the reaction mixture, asdescribed in section (c) showed complete conversion to the product 4feluting at Rt=14.5 minutes. The product was purified by preparative HPLCas described in section (d). Yield=22 mg (59%); MALDI-TOF MS 1565.8observed.

B4-AE-NHS, compound 4h. A solution of compound 4f (22 mg, 14 umoles) inanhydrous methanol (2 mL) was treated with diisopropylethylamine (12.3uL, 70 umoles) and glutaric anhydride (8 mg, 70 mmoles). The reactionwas stirred at room temperature. After 30 minutes, HPLC analysis, asdescribed in section (d), showed complete conversion to the glutaratederivative 4g eluting at Rt=15.9 minutes. The reaction mixture wasdiluted with anhydrous toluene (3 mL) and concentrated under reducedpressure.

The crude product was dissolved in anhydrous DMF (2 mL) and treated withdiisopropylethylamine (24.6 uL, 0.14 mmol) and TSTU (42 mg, 0.14 mmol).After stirring for 30 minutes, HPLC analysis, as described in section(c), showed >70% conversion to the product 4h eluting at Rt=16.9minutes. The product was purified by preparative HPLC as described insection (d). The HPLC fractions containing product were frozen at −80°C. and lyophilized to dryness. The lyophilized product was dissolved inanhydrous MeCN and transferred to a tared round bottom flask andconcentrated under reduced pressure. Yield=18.8 mg (75%); MALDI-TOF MS1776.5 observed.

The following reactions describe the synthesis of B4-AE-NHS, 4h.

EXAMPLE 5 B04-AE-NHS, 5i

a) Compound 5b. 1,3-Dimethoxy-2-propanol, 5a, was synthesized asdescribed by Kang et al. in Bull. Korean Chem. Soc. 2006, 27, 1364-1370.Crude 1,3-dimethoxy-2-propanol (10.66 g, 0.089 mol) and potassiumhydroxide (3 g, 0.00534 mol) was stirred at 80° C. under a nitrogenatmosphere until all the potassium hydroxide dissolved. Epichlorohydrin(1.65 g, 0.00178 mol) was then added dropwise and the reaction washeated at 100° C. under a nitrogen atmosphere for 24 hours. The reactionwas then cooled to room temperature and partitioned between ethylacetate (75 mL) and saturated ammonium chloride solution (75 mL). Theethyl acetate layer was separated and the aqueous layer was extractedonce more with ethyl acetate (50 mL). The combined ethyl acetateextracts were dried over anhydrous magnesium sulfate and concentratedunder reduced pressure. The recovered light brown oil (3.7 g) was used asuch in the next reaction.

b) Compound 5c. Compound 5b (3.7 g, 0.0125 mol) was dissolved ananhydrous pyridine (15 mL) and treated with 4-dimethylpyridine (0.381 g,3.1 mmol) and p-toluenesulfonyl chloride (4.8 g, 0.0025 mol). Thereaction was stirred at room temperature under a nitrogen atmosphere for3 days. The solvent was then removed under reduced pressure and theresidue was partitioned between ethyl acetate (75 mL) and 1N HCl (50mL). The ethyl acetate layer was separated and washed with 2% sodiumhydroxide solution (50 mL) and saturated ammonium chloride solution (50mL). It was then dried over anhydrous magnesium sulfate and concentratedunder reduced pressure. The crude product (6.6 g) was purified by flashchromatography on silica gel using 75:24:1; hexanes:ethyl acetate:methanol as eluent. Yield=1.73 g, light yellow oil.

c) Compound 5d. A mixture of 2,7-dihydroxy acridine methyl ester, 1c(0.1 g, 0.24 mmol) compound 5c (0.54 g, 1.2 mmol) and cesium carbonate(0.2 g, 0.06 mmol) in anhydrous DMF (5 mL) was heated at 100° C. under anitrogen atmosphere for 4-5 hours. A small portion of the reactionmixture was then analyzed by HPLC using a Phenomenex, C₁₈ 4.6 mm×25 cmcolumn and a 30 minute gradient of 10→100% B (A=water with 0.05% TFA,B=MeCN with 0.05% TFA) at a flow rate of 1.0 mL/minute and UV detectionat 260 nm. Product was observed eluting at Rt=26.2 minutes and was themajor component. The reaction was then cooled to room temperature andconcentrated under reduced pressure. The residue was partitioned betweenethyl acetate (75 mL) and water (50 mL). The ethyl acetate layer wasseparated, dried over anhydrous magnesium sulfate and concentrated underreduced pressure. The crude product (0.45 g) was purified by preparativeTLC on silica gel using 1% methanol in ethyl acetate as eluent. Yield=64g (28%); MALDI-TOF MS 973.8 observed.

d) Compound 5f A mixture of compound 5d (64 mg, 65.7 umoles), distilled1,3-propane sultone (1.6 g, 13.1 mmol) and sodium bicarbonate (110 mg,1.3 mmol) was heated at 150° C. under a nitrogen atmosphere. After 2hours, a small portion was withdrawn, diluted with methanol and analyzedby HPLC using a Phenomenex, C₁₈ 4.6 mm×25 cm column and a 30 minutegradient of 10→100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) ata flow rate of 1.0 mL/minute and UV detection at 260 nm. Product wasobserved eluting at Rt=20.5 minutes (>60% conversion). The reaction wascooled to room temperature and 20 mL of 1:1, ethyl acetate/hexanes wasadded. After brief sonication to disperse the gummy product, the solventwas decanted and the product 5e was dried under vacuum.

The crude acridinium ester 5e was suspended in IN HCl (10 mL) andrefluxed under a nitrogen atmosphere for 2 hours. HPLC analysis, asdescribed above, indicated complete conversion to product 5f eluting at17.5 minutes. The product was purified by preparative HPLC using an YMC,C₁₈ 30×300 mm column and the same gradient at described above at asolvent flow rate of 20 mL/minute and UV detection at 260 nm. The HPLCfractions containing product were combined and concentrated underreduced pressure. Yield=12 mg (17%); MALDI-TOF MS 1082.4 observed.

e) Compound 5g. A solution of compound 5f (12 mg, 11.1 umoles) inanhydrous DMF (1 mL) was treated with diisopropylethylamine (4.0 uL, 22umoles) and TSTU (5 mg, 16.7 umoles). The reaction was stirred at roomtemperature. After 30 minutes, HPLC analysis, as described in section(c) showed complete conversion to the NHS ester eluting at Rt=19.5minutes. This reaction was added dropwise to a stirred solution of2,2′-(ethylenedioxy)bis(ethylamine) (16 ul, 0.11mmol) in anhydrous DMF(1.0 mL). After one hour, HPLC analysis of the reaction mixture, asdescribed in section (c) showed complete conversion to the product 5geluting at Rt=14.7 minutes. The product was purified by preparative HPLCas described in section (d). Yield=15.4 mg (quantitative); MALDI-TOF MS1212.9 observed.

B04-AE-NHS, compound 5i. A solution of compound 5g (15.4 mg, 12.7umoles) in anhydrous methanol (2 mL) was treated withdiisopropylethylamine (11 uL, 63.5 umoles) and glutaric anhydride (7.2mg, 63.5 umoles). The reaction was stirred at room temperature. After 30minutes, HPLC analysis, as described in section (c), showed completeconversion to the glutarate derivative 5h eluting at Rt=16.2 minutes.The reaction mixture was diluted with anhydrous toluene (3 mL) andconcentrated under reduced pressure. The crude product was dissolved inanhydrous DMF (2 mL) and treated with diisopropylethylamine (22 uL,0.126 mmol) and TSTU (38 mg, 0.126 mmol). After stirring for 15 minutes,HPLC analysis, as described in section (c), showed >80% conversion tothe product 5i eluting at Rt=17.2 minutes. The product was purified bypreparative HPLC as described in section (d). The HPLC fractionscontaining product were frozen at −80° C. and lyophilized to dryness.The lyophilized product was dissolved in anhydrous MeCN and transferredto a tared round bottom flask and concentrated under reduced pressure.Yield=12.3 mg (68%); MALDI-TOF MS 1423.8 observed.

The following reactions describe the synthesis of B04-AE-NHS, compound5i

EXAMPLE 6 B1-AE-E2, 6b

a) Compound 6a. A solution of compound 1f (26 mg, 28.7 umoles) inanhydrous DMF (2 mL) was treated with diisopropylethylamine (7.5 uL, 43umoles) and TSTU (10.4 mg, 35 umoles). The reaction was stirred at roomtemperature. After 30 minutes, HPLC analysis of the reaction mixtureusing a Phenomenex, C₁₈ 4.6 mm×25 cm column and a 30 minute gradient of10→100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at a flow rateof 1.0 mL/minute and UV detection at 260 nm, indicated completeconversion to the NHS ester eluting at Rt=18.2 minutes. This reactionwas added dropwise to a stirred solution of diamino hexa(ethylene)glycol (U.S. Pat. No. 6,664,043), (40 mg, 0.142 mmol) in anhydrous DMF(2.0 mL). After 30 minutes, HPLC analysis of the reaction mixture showedcomplete conversion to the product 6a eluting at Rt=14.1 minutes. Theproduct was purified by preparative HPLC using an YMC, C₁₈ 30×300 mmcolumn and the same gradient as described above at a solvent flow rateof 20 mL/minute and UV detection at 260 nm. The HPLC fractionscontaining product were combined and concentrated under reducedpressure. Yield=26 mg (78%); MALDI-TOF MS 1168.6 observed.

b) B1-AE-E2, 6b. Estradiol-6-carboxymethyloxime (1 mg, 2.78 umoles) inDMF (0.1 mL) was combined with compound 6a (3.25 mg, 2.78 umoles) andtreated with diisopropylethylamine (1 uL, 5.56 umoles) followed by BOPreagent (1.84 mg, 4.17 umoles) added as a solution in DMF (0.184 mL of a10 mg/mL solution). The reaction was stirred at room temperature 2 h.HPLC analysis, as described in section (a), indicated >80% conversion tothe product eluting at Rt=18.2 minutes. The product was purified bypreparative HPLC using an YMC, C₁₈ 20×250 mm column and a 30 minute of10→70% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) gradient of ata solvent flow rate of 16 mL/minute and UV detection at 260 nm. The HPLCfractions containing product were combined, frozen at −80oC andlyophilized to dryness. Yield=2.8 mg (67%); MALDI-TOF MS 1511.2observed.

The following reactions describe the synthesis of B1-AE-E2, 6b.

EXAMPLE 7 B2-AE-E2, 7b

a) Compound 7a. A solution of compound 2e (30 mg, 28 umoles) inanhydrous DMF (2 mL) was treated with diisopropylethylamine (7.2 uL, 42umoles) and TSTU (10 mg, 34 umoles). The reaction was stirred at roomtemperature. After 30 minutes, HPLC analysis of the reaction mixtureusing a Phenomenex, C₁₈ 4.6 mm×25 cm column and a 30 minute gradient of10→100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at a flow rateof 1.0 mL/minute and UV detection at 260 nm, indicated completeconversion to the NHS ester eluting at Rt=17.7 minutes. This reactionwas added dropwise to a stirred solution of diamino hexa(ethylene)glycol (U.S. Pat. No. 6,664,043), (40 mg, 0.142 mmol) in anhydrous DMF(1.0 mL). After 30 minutes, HPLC analysis of the reaction mixture showedcomplete conversion to the product 7a eluting at Rt=14 minutes. Theproduct was purified by preparative HPLC using an YMC, C₁₈ 30×300 mmcolumn and the same gradient as described above at a solvent flow rateof 20 mL/minute and UV detection at 260 nm. The HPLC fractionscontaining product were combined and concentrated under reducedpressure. Yield=28.3 mg (76%); MALDI-TOF MS 1345.4 observed.

b) B2-AE-E2, 7b. Estradiol-6-carboxymethyloxime (1 mg, 2.78 umoles) inDMF (0.1 mL) was combined with compound 7a (3.74 mg, 2.78 umoles) andtreated with diisopropylethylamine (1 uL, 5.56 umoles) followed by BOPreagent (1.84 mg, 4.17 umoles) added as a solution in DMF (0.184 mL of a10 mg/mL solution). The reaction was stirred at room temperature 2 h.HPLC analysis, as described in section (a), indicated >80% conversion tothe product eluting at Rt=18.1 minutes. The product was purified bypreparative HPLC using an YMC, C₁₈ 20×250 mm column and a 30 minute of10→70% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) gradient of ata solvent flow rate of 16 mL/minute and UV detection at 260 nm. The HPLCfractions containing product were combined, frozen at −80oC andlyophilized to dryness. Yield=4.6 mg (98%); MALDI-TOF MS 1688.4observed.

The following reactions describe the synthesis of B2-AE-E2, 7b.

EXAMPLE 8 B4-AE-E2, 8b

a) Compound 8a. A solution of compound 4e (26 mg, 18 umoles) inanhydrous DMF (2 mL) was treated with diisopropylethylamine (4.0 uL, 27umoles) and TSTU (6.6 mg, 22 umoles). The reaction was stirred at roomtemperature. After 30 minutes, HPLC analysis of the reaction mixtureusing a Phenomenex, C₁₈ 4.6 mm×25 cm column and a 30 minute gradient of10→100% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) at a flow rateof 1.0 mL/minute and UV detection at 260 nm, indicated completeconversion to the NHS ester eluting at Rt=18.7 minutes. This reactionwas added dropwise to a stirred solution of diamino hexa(ethylene)glycol (U.S. Pat. No. 6,664,043), (25 mg, 0.089 mmol) in anhydrous DMF(2.0 mL). After 30 minutes, HPLC analysis of the reaction mixture showedcomplete conversion to the product 8a eluting at Rt=15.1 minutes. Theproduct was purified by preparative HPLC using an YMC, C₁₈ 30×300 mmcolumn and the same gradient as described above at a solvent flow rateof 20 mL/minute and UV detection at 260 nm. The HPLC fractionscontaining product were combined and concentrated under reducedpressure. Yield=22.5 mg (73%); MALDI-TOF MS 1698.6 observed.

b) B4-AE-E2, 8b. Estradiol-6-carboxymethyloxime (1 mg, 2.78 umoles) inDMF (0.1 mL) was combined with compound 8a (4.72 mg, 2.78 umoles) andtreated with diisopropylethylamine (1 uL, 5.56 umoles) followed by BOPreagent (1.84 mg, 4.17 umoles) added as a solution in DMF (0.184 mL of a10 mg/mL solution). The reaction was stirred at room temperature 2 h.HPLC analysis, as described in section (a), indicated >80% conversion tothe product eluting at Rt=18.9 minutes. The product was purified bypreparative HPLC using an YMC, C₁₈ 20×250 mm column and a 30 minute of10→70% B (A=water with 0.05% TFA, B=MeCN with 0.05% TFA) gradient of ata solvent flow rate of 16 mL/minute and UV detection at 260 nm. The HPLCfractions containing product were combined, frozen at −80oC andlyophilized to dryness. Yield=4.0 mg (70%); MALDI-TOF MS 2040.9observed.

The following reactions describe the synthesis of B4-AE-E2, 8b.

EXAMPLE 9

General procedure for labeling anti-TSH Mab with acridinium ester. Astock solution of the antibody (5 mg/mL, 50 uL, 0.5 mg, 3.4 nmoles) wasdiluted with either 0.1 M phosphate buffer pH 8 (150 uL) or 0.1 M sodiumcarbonate pH 9 (150 uL) to give a 2.5 mg/mL solution. To this solutionwas added 20 equivalents of the acridinium NHS ester as a DMF solution.For example, using B1-AE-NHS, this entailed the addition of 83 ug addedas 8.3 uL of a 10 mg/mL DMF solution of the acridinium ester.

The labeling reactions were stirred gently at room temperature for 3-4hours and were then diluted with de-ionized water (1.8 mL). Thesediluted solutions were then transferred to 2 mL Centricon filters (MW30,000 cutoff) and centrifuged at 4500 G to reduce the volume to ˜0.2mL. This process was repeated three more times. The filtered conjugateswere finally diluted into a total volume of 200 uL de-ionized water formass spectral analysis and RLU measurements.

Mass spectra were recorded on a Voyager DE MALDI-TOF mass spectrometerand the unlabeled antibody was used as the reference. Approximately 2 uLof the conjugate solution was mixed with 2 uL of sinnapinic acid matrixsolution (HP) and the spotted on a MALDI plate. After complete drying,mass spectra were recorded. From the difference in mass values for theunlabeled antibody and the conjugates, the extent of AE incorporationcould be measured. Typically, under these labeling conditions, 3-6 AElabels were incorporated in the antibody.

EXAMPLE 10

Measurement of Stability. Maximization of stability of acridinium estersis one parameter by which assay precision is enhanced. Chemiluminescencestability of several acridinium esters covalently attached to anti-TSHantibody were analyzed for the correlation of molecular structure of theacridinium ester to the stabilty of chemiluminescent under both anominal storage temperature of 4° C. and an elevated storage temperatureof 37° C. Equivalent amounts of acridinium ester-labeled antiTSH(thyroid stimulating hormone) antibody each conjugated to a differentacridinium ester were diluted to a concentration of 0.2 nanomolar inSiemens Healthcare Diagnostics TSH3 (thyroid stimulating hormone) LiteReagent buffer consisting of 0.1 M sodium N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonate (HEPES), 0.15 M sodium chloride, 7.7 mMsodium azide, 1.0 mM tetrasodium ethylenediaminetetraacetate, (EDTA), 12mM t-octylphenoxypolyethoxyethanol (Triton X-100), 76 uM bovine serumalbumin (BSA), 7 uM mouse immunoglobin (IgG), pH 7.7. Each acridiniumester solution was partitioned into two sets of storage vessels. One setof storage vessels was kept at 4° C. and the other at 37° C. Startingfrom the day of initial dilution the chemiluminescence from 10microliters of each acridinium ester-antibody solution was determinedunder standard conditions on a Berthold Technolgies Autolumat LB953luminometer with sequeuntial addition of 300 microliters each of SiemensHealthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid and 0.5%hydrogenperoxide) and Siemens Healthcare Diagnostics Flash Reagent 2(0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride).

EXAMPLE 11

Measurement of Fractional Non-specific Binding. Minimization offractional nonspecific binding (fNSB) of acridinium esters to a solidphase is one parameter by which assay sensitivity is enhanced. Thefractional nonspecific bindings of several acridinium esters covalentlyattached to antiTSH antibody were analyzed for correlation to themolecular structure of the acridinium ester. Equivalent amounts ofacridinium ester-labeled antiTSH (thyroid stimulating hormone) antibodyeach conjugated to a different acridinium ester were diluted to aconcentration of 2 nanomolar in Siemens Healthcare Diagnostics TSH3(thyroid stimulating hormone) Lite Reagent buffer consisting of 0.1 Msodium N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonate (HEPES), 0.15M sodium chloride, 7.7 mM sodium azide, 1.0 mM tetrasodiumethylenediaminetetraacetate, (EDTA), 12 mMt-octylphenoxypolyethoxyethanol (Triton X-100), 76 uM bovine serumalbumin (BSA), 7 uM mouse immunoglobin (IgG), pH 7.7. Following dilution100 microliters of the acridinium ester containing solutions were eachwith 200 microliters of horse serum (Siemens Healthcare DiagnosticsMulti-diluent 1) and 200 microliters of either of two solid phases. Thefirst solid phase was 200 microliters of Siemens Healthcare DiagnosticsACS PTH (parathyroid hormone) Solid Phase containing 50 micrograms ofmagnetic latex microparticles (MLP) derivatized with antiPTH antibody.The second solid phase was 200 microliters of Siemens HealthcareDiagnostics ACS TSH3 (thyroid stimulating hormone) Solid Phasecontaining 60 micrograms of paramagnetic microparticles (PMP)derivatized with antiTSH antibody. The particles were magneticallycollected and washed twice with water after an incubation of 10 minutesto allow interaction between the acridinium ester labeled antbodies andthe solid phases. The chemiluminescence of acridinium ester associatedwith the particles was meaured under standard conditions on a BertholdTechnolgies Autolumat LB953 luminometer with sequeuntial addition of 300microliters each of Siemens Healthcare Diagnostics Flash Reagent 1 (0.1M nitric acid and 0.5% hydrogenperoxide) and Siemens HealthcareDiagnostics Flash Reagent 2 (0.25 M sodium hydroxide and 0.05%cetyltrimethylammonium chloride). Chemiluminescence was measured for 5.0seconds. Fractional nonspecific binding (fNSB) is calculated as theratio of particle-bound chemiluminescence to total chemiluminescenceinput. In general hydrophobicity of an acridinium ester elevates fNSBand is undesirable when distinguishing small amounts of specific signal,conversely hydrophilicity of an acridinium ester lowers fNSB and isdesirable when distinguishing small amounts of specific signal.

EXAMPLE 12

Measurement of Chemiluminescence Kinetics. Hastening of acridinium esterchemiluminescence rates is one parameter by which assay throughput ratescan be increased. Chemiluminescence kinetics of several acridiniumesters covalently attached to anti-TSH antibody were analyzed for thecorrelation of molecular structure of the acridinium ester to its rateof chemiluminescence light emission. Each acridinium ester labeledantibody was diluted to a concentration of 0.2 nanomolar in a bufferconsisting of 0.1 M sodium phosphate, 0.15 M sodium chloride, 6 mMsodium azide and 1 g/L bovine serum albumin (BSA). The chemiluminescencekinetics for 10 microliters of each acridinium ester-antibody conjugatetested was integrated in 0.1 second intervals for 20 seconds understandard conditions on a Berthold Technolgies Autolumat LB953luminometer with sequeuntial addition of 300 microliters each of SiemensHealthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid and 0.5%hydrogenperoxide) and Siemens Healthcare Diagnostics Flash Reagent 2(0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride). Thechemiluminescence kinetics of the tested acridinium esters were comparedfor relative rate of light emission.

EXAMPLE 13

Measurement of Quantum Yield. Increasing acridinium esterchemiluminescence quantum yield is one parameter by which assaysensitivity can be increased. Chemiluminescence quantum yields ofseveral acridinium esters covalently attached to antiTSH antibody weretested for the correlation of molecular structure of the acridiniumesters to the magnitude of their chemiluminescence light output. Eachacridinium ester labeled antibody was diluted to a concentration of 0.2nanomolar in a buffer consisting of 0.1 M sodium phosphate, 0.15 Msodium chloride, 6 mM sodium azide and 1 g/L bovine serum albumin (BSA).The chemiluminescence kinetics for 10 microliters of each acridiniumester-antibody conjugate tested was measured for 10 seconds understandard conditions on a Berthold Technolgies Autolumat LB953luminometer with sequeuntial addition of 300 microliters each of SiemensHealthcare Diagnostics Flash Reagent 1 (0.1 M nitric acid and 0.5%hydrogenperoxide) and Siemens Healthcare Diagnostics Flash Reagent 2(0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride). Thechemiluminescence quantum yield was calculated as the ratio of thechemiluminescence to the amount of acridinium ester tested.

All patent and non-patent literature referenced in this specification ishereby incorporated by reference.

The invention having been described by the foregoing description of thepreferred embodiments, it will be understood that the skilled artisanmay make modifications and variations of these embodiments withoutdeparting from the spirit or scope of the invention as set forth in thefollowing claims.

We claim:
 1. A hydrophilic, high quantum yield acridinium ester havingthe following structure:

wherein, R₁ is a methyl or sulfopropyl group; G is a branched groupindependently selected at each occurrence from:

where R₂, R_(3,) R₄, R₅, R₆ and R₇ are independently at each occurrencea methyl group or a group —(CH₂CH₂O)_(n)CH₃, where n is an integer from1 to 5; and R₁₂ is an electrophilic or nucleophilic group forconjugating the acridinium compound to an analyte, an analyte analog, ora binding molecule for an analyte.
 2. An acridinium ester according toclaim 1 wherein G is, at one or both occurrences, a group:

where R₂ and R₃ are independently at each occurrence a methyl group or agroup —(CH₂CH₂O)_(n)CH₃, where n is an integer from 1 to
 5. 3. Anacridinium ester according to claim 2 wherein G is a group:

at one or both occurrences.
 4. An acridinium ester according to claim 2wherein G is a group:

at one or both occurrences.
 5. An acridinium ester according to claim 2wherein G is a group:

at one or both occurrences.
 6. An acridinium ester according to claim 1wherein G is, at one or both occurrences, a group:

where R₄, R₅, R₆ and R₇ are independently at each occurrence a methylgroup or a group —(CH₂CH₂O)_(n)CH₃, where n is an integer from 1 to 5.7. An acridinium ester according to claim 6 wherein G is, at one or bothoccurrences, a group:

at one or both occurrences.
 8. An acridinium ester according to claim 6wherein G is a group:

at one or both occurrences.
 9. An acridinium ester according to claim 1,where R₁₂ is selected from the group consisting of: (1) —OH; (2)—O—N-succinimidyl; (3) —NH—(CH₂)₅—C(O)—O—N-succinimidyl; (4)—NH—(CH₂)₅—COOH; (5)—NH—(C₂H₄O)_(n)—C₂H₄NH—C(O)—(CH₂)₃—C(O)—O—N-succinimidyl wherein n=1 to5; (6) —NH—(C₂H₄O)_(n)—C₂H₄NH—C(O)—(CH₂)₃—COOH, wherein n=1 to 5; (7)—NH—(C₂H₄O)_(n)—C₂H₄NH₂, wherein n=1 to 5; and (8) —NH—R—NHR, wherein Ris independently hydrogen, alkyl, alkenyl, alkynyl, or aralkyl; whereinR optionally comprises up to 20 heteroatoms.
 10. An acridinium esteraccording to claim 9, wherein R₁₂ is —OH.
 11. An acridinium esteraccording to claim 9, wherein R₁₂ is —NH—(C₂H₄O)_(n)—C₂H₄NH₂, whereinn=1 to
 5. 12. An acridinium ester according to claim 9, wherein R₁₂ is:—NH—(C₂H₄O)_(n)—C₂H₄NH—C(O)—(CH₂)₃—C(O)—O—R″ wherein n=1 to 5; and whereR″ is hydrogen or —N-succinimidyl.
 13. An acridinium ester according toclaim 1, having the following structure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.
 14. An acridinium ester according to claim 1having the following structure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.
 15. An acridinium ester according to claim 1having the following structure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.
 16. An acridinium ester according to claim 1having the following structure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.
 17. An acridinium ester according to claim 1having the following structure:

where R₁₂ is an electrophilic or nucleophilic group for conjugating theacridinium compound to an analyte, an analyte analog, or a bindingmolecule for an analyte.
 18. The acridinium ester according to any ofclaims 14-18, wherein R₁₂ is —OH.
 19. An assay for the detection orquantification of an analyte comprising the steps of: (a) providing aconjugate comprising: (i) a binding molecule specific for an analyte;and (ii) a hydrophilic, high quantum yield and fast light emittingacridinium ester according to claim 1; (b) providing a solid supporthaving immobilized thereon a second binding molecule specific for saidanalyte; (c) mixing the conjugate, the solid phase and a samplesuspected of containing the analyte to form a binding complex; (d)separating the binding complex captured on the solid support; (e)triggering chemiluminescence of the binding complex from step (d) byadding chemiluminescence triggering reagents; (f) measuring the amountof light emission with a luminometer; and (g) detecting the presence orcalculating the concentration of the analyte by comparing the amount oflight emitted from the reaction mixture with a standard dose responsecurve which relates the amount of light emitted to a known concentrationof the analyte.
 20. An assay for the detection or quantification of ananalyte comprising the steps of: (a) providing a conjugate of an analytewith a hydrophilic, high quantum yield and fast light emittingacridinium ester according to claim 1; (b) providing a solid supportimmobilized with a binding molecule specific for the analyte; (c) mixingthe conjugate, solid support and a sample suspected of containing theanalyte to form a binding complex; (d) separating the binding complexcaptured on the solid support; (e) triggering the chemiluminescence ofthe binding complex from step (d) by adding chemiluminescence triggeringreagents; (f) measuring the amount of light with an luminometer; and (g)detecting the presence or calculating the concentration of the analyteby comparing the amount of light emitted from the reaction mixture witha standard dose response curve which relates the amount of light emittedto a known concentration of the analyte.